WO1992013264A1

WO1992013264A1 - Method of measuring luminescence in an assay by luminescence

Info

Publication number: WO1992013264A1
Application number: PCT/FR1992/000069
Authority: WO
Inventors: Michel Mabile; Gérard Mathis; Etienne Jean-Pierre Jolu; Dominique Pouyat; Christophe Dumont
Original assignee: Cis Bio International
Priority date: 1991-01-28
Filing date: 1992-01-27
Publication date: 1992-08-06
Also published as: FR2672128A1; EP0569496B1; US5527684A; DE69200445T2; ATE112055T1; DE69200445D1; AU1274292A; JP3233285B2; FR2672128B1; ES2066602T3; EP0569496A1; DK0569496T3; JPH06508919A

Abstract

A method for measuring luminescence in an assay by luminescence is described wherein certain disturbances due to the measuring medium can be corrected. The method is characterized in that it employs at least one luminescent tracer compound and a luminescent compound used as an internal reference which, when exposed to the same excitation wavelength, are capable of emission at different wavelengths, μ2 et μ1 respectively, either by direct luminescence or by induction of luminescent emission, and that the measurement of tracer compound luminescence at wavelength μ2 is corrected on the basis of the measurement of reference compound luminescence at wavelength μ1. The use of the method in a homogeneous analyte detection and/or determination process, and a device for its implementation, are also described.

Description

Method for measuring the luminescence emitted in a luminescence assay.

The subject of the invention is a method for measuring the luminescence emitted in a luminescence assay making it possible to correct certain disturbances due to the measurement medium.

The use of immunoassays for the qualitative and quantitative analysis of compounds in biological fluids is currently widespread.

Among the existing techniques, fluorimetry assays have become increasingly important.

Indeed, they have a certain number of advantages among which the sensitivity, the speed of the measurement, the stability and the harmlessness of the reagents marked by fluorescent compounds and the relatively reduced cost.

It is known that detection methods using fluorescence are intrinsically very sensitive and could allow detection limits lower than those reached by immunoassays using radiolabelled reagents, in particular by the use of modular laser light sources (I. Wieder , Immunofluorescence and related staining techniques, 1978, Elsevier).

Many fluorescent molecules which can be used as a tracer in this type of assay have been previously described; among these, mention may in particular be made of rare earth complexes which have interesting properties.

By "tracer" is meant either a luminescent molecule emitting direct luminescence, or a luminescent molecule capable of inducing luminescence emission, said molecule being capable of being coupled to one of the reagents of the assay, and the emission luminescence, direct or induced, allowing the detection and / or determination of the desired analyte.

The use of particular complexes, the rare earth cryptates, is described for example in applications EP 0 321 353, 0 180 492, 0 232 348 or the international application W0 90/04791. These rare earth cryptates have the advantage of being very stable in protein and saline medium, this property being particularly important in the case of homogeneous immunoassays.

The sensitivity of the measurement is nevertheless limited by various interfering parameters, among which we can cite:

the spectroscopic properties of the medium, and in particular its intrinsic fluorescence, due in particular to the parasitic emissions of the molecules present in the measurement medium and likely to be excited and to emit at wavelengths close to those of the fluorescent tracer and / or with high intensities; its absorption, which results in a loss of exciting light; its light scattering properties when the measurement medium is not clear,

- quenching the fluorescence emitted ("quenching") by inhibitors present in the medium,

- the composition of the device, and in particular the stray reflections caused by the device,

All of these interferences considerably affect the sensitivity and reproducibility of the measurement.

Some of these problems have already been solved by various techniques.

In particular, the time-resolved fluorescence measurement methods make it possible to partially remedy the parasitic emissions (background noise). The principle of these methods resides in the fact that the measurement of the fluorescence emitted by a tracer molecule having a relatively long emission lifetime is carried out, the measurement being delayed in time beyond the lifetime of d emission of other molecules present.

In this case, it is necessary to use fluorescent tracer molecules with a relatively long lifetime, such as chelates and rare earth cryptates.

However, no satisfactory solution has been provided to the limitations due to the spectroscopic properties of the medium, and in particular to its absorption.

Indeed, among the techniques proposed to avoid the filter effect of the medium, none allows both easy implementation, inexpensive as well as obtaining a high sensitivity and very good reproducibility in the measurement.

In particular, the solution consisting in strongly diluting the sample is unfavorable to the sensitivity of the detection.

In addition, the implementation of a double excitation beam system entails the use of expensive equipment and special measuring cuvettes, which are difficult to standardize. In addition, the systematic measurement of the absorption of the medium before the measurement of the fluorescence of the sample increases the assay process.

Application EP 355 849 describes a method and an automatic apparatus making it possible to verify the reliability of the fluorescence measurement of a sample and to correct this measurement with respect to an internal reference. This requires prior to the measurement of the test sample, to carry out a measurement with 2 reference samples, one "blank" containing only the measurement medium and the other containing the measurement medium and the fluorescent marker.

Application EP 91 126 relates to a fluorimeter making it possible to measure, in parallel with the fluorescence of the sample, the transmission of the latter and the fluctuations of the excitation energy, in order to correct the measured fluorescence. This system requires a special measuring cell allowing the excitation beam to pass, because the transmission must be measured in alignment with the excitation beam.

Other systems implement a system for dividing the excitation beam, as described for example in applications EP 174 722 and EP 241 268.

The subject of the invention is therefore a method for measuring the luminescence emitted in a luminescence assay, characterized in that at least one luminescent tracer compound and one luminescent compound used as internal reference are used which, when they are subjected to the same excitation wavelength, are likely to emit either by direct luminescence, or by induction of an emission of luminescence, at different wavelengths, respectively λ ₂ and λ ₁ , the luminescence of the reference compound reflecting the optical quality of the medium, and in that which is corrected measuring the luminescence emitted by the tracer compound at the wavelength λ ₂ by measuring the luminescence emitted by the reference compound at the wavelength λ ₁ .

By "reference compound" is meant either a luminescent molecule emitting direct luminescence, or a luminescent molecule capable of inducing luminescence emission, said luminescence emission, direct or indirect, not being disturbed by the reactive system of the assay .

The luminescent compounds which can be used in the measurement method according to the invention can either emit directly at their emission wavelength or at another wavelength, for example in the case of a spectrum shift linked to the reactive system dosage.

The luminescent compounds which can be used in the measurement method according to the invention can optionally emit indirectly by inducing an emission of luminescence, as in particular in the case of homogeneous methods by energy transfer.

Advantageously, the luminescence emissions at wavelengths λ ₂ and λ ₁ are detected simultaneously.

The method according to the invention, which uses a single excitation beam, makes it possible to carry out easily and reliably the measurement of the luminescence emitted in a luminescence assay without requiring complex apparatus, by eliminating the disturbances due spectroscopic properties of the assay medium.

Advantageously, the emission wavelengths of the luminescent tracer compound and of the luminescent compound of reference λ ₁ and λ ₂ will be different but preferably close (by having a difference for example less than or equal to 100 nm) so as to that the disturbance of the luminescence emission due to the absorption of the medium occurs in the same way manner vis-à-vis the emission of the tracer compound and that of the reference compound.

It will be noted that, advantageously, the method according to the present invention does not require that the sample to be assayed be placed in a particular measuring tank.

The luminescence emission of the reference compound at wavelength λ ₁ makes it possible to correct the measurement carried out at wavelength λ ₂ . The correction can be carried out for example, by dividing this last measurement by the measurement carried out at the wavelength λ ₁ . Other correction means can be used, such as, for example, a correction method integrated into the apparatus in which a counting rate is fixed on the channel measuring the luminescence emission of the reference compound at the length of wave λ ₁ . When this rate is reached, the end of the measurement is triggered on the channel measuring the emission of luminescence at the wavelength λ ₂ . The value obtained on this channel is therefore directly corrected.

Other correction methods known to those skilled in the art can also be used.

In a preferred aspect, the subject of the invention is a method for measuring the fluorescence emitted in an assay by fluorometry, characterized in that at least one fluorescent tracer compound and a fluorescent compound used as internal reference are used, which when subjected to the same excitation wavelength are capable of emitting, either by direct fluorescence, or by induction of a fluorescence emission, at different wavelengths, λ ₂ and λ _{1 respectively} , the fluorescence of the reference compound reflecting the optical quality of the medium, and the measurement of the fluorescence emitted by the tracer compound then being corrected by the measurement of the fluorescence emitted by the reference compound.

The method according to the invention makes it possible to achieve a measurement sensitivity of the order of a picomole / liter, while the phenomena mentioned above usually limit the sensitivity of the assay, especially during homogeneous assays in serum medium, at analyte concentrations of the order of a micromole / liter.

In an advantageous aspect of the invention, the luminescent tracer compound and the reference compound are one and the same compound.

This first variant of the method according to the invention preferably applies when using a homogeneous method of detection and / or determination by luminescence of an analyte in a medium capable of containing it in which the measurement of the emitted luminescence representative of the amount of analyte in the medium is carried out at an emission wavelength different from that of the tracer compound.

For example, this case arises when the emitted luminescence representative of the analyte results from an energy transfer between a donor luminescent compound and an accepting luminescent compound, the latter emitting at a wavelength λ ₂ while the compound donor also serving as a reference compound emits at a wavelength λ ₁ .

In particular, this case also occurs when the tracer compound emits at different wavelengths λ ₁ and λ ₂ depending on whether it is, respectively, not engaged or engaged in the reactive assay system.

By "homogeneous process" is meant a dosing process in which the measurement does not require the prior separation of the components of the dosage.

Surprisingly, it has indeed been found that the intensity of the signal emitted by the luminescent compound of reference at the wavelength λ ₁ is practically constant. The signal emitted therefore depends only on the optical properties of the medium in which the assay is carried out and not on the amount of analyte, and can serve as a reference.

In the case of an emission of luminescence resulting from an energy transfer, the signal reflecting both the amount of analyte to be assayed and the optical properties of the measurement medium is detected at a wavelength λ ₂ and corrected by the measurement made at the wavelength λ ₁ . Preferably, this first variant of the method according to the invention will be used in a homogeneous method of detection and / or determination by luminescence of an analyte in a medium capable of containing it using an excess method consisting of:

1) adding, to said medium containing the desired analyte, a first reagent consisting of at least one receptor of said analyte, coupled with a luminescent donor compound,

2) adding a second reagent consisting of one or more other receptors of said analyte, said second reagent being coupled with a luminescent acceptor compound,

3) incubating said medium after each addition of reagents or after the addition of the two reagents,

4) to excite the resulting medium at the excitation wavelength of the luminescent donor compound

5) measuring the signal of the donor luminescent compound at a wavelength λ ₁ , this measurement serving as a reference, and the signal resulting from the transfer of energy at a different wavelength λ ₂ .

In an advantageous aspect, the first reagent and the second reagent used in the methods of detection and / or determination by luminescence of an analyte indicated above are added simultaneously to the medium containing the analyte sought.

In another aspect of the invention, this variant of the method can be used in a homogeneous method of detection and / or determination by luminescence of an analyte in a medium capable of containing it using a competitive method. consisting of:

2) adding a second reagent consisting of the analyte coupled with a luminescent acceptor compound,

3) incubating said medium after each addition of reagents or after the addition of the two reagents. 4) to excite the resulting medium at the excitation wavelength of the luminescent donor compound,

This variant of the process can also advantageously be used in a homogeneous process for the detection and / or determination by luminescence of an analyte in a medium capable of containing it using a competition method consisting of:

1) adding, to said medium containing the desired analyte, a first reagent consisting of at least one receptor of said analyte, coupled with a luminescent acceptor compound,

2) adding a second reagent consisting of the analyte coupled with a luminescent donor compound,

4) to excite the resulting medium at the excitation wavelength of the luminescent donor compound,

In another advantageous use of the method of the invention, at least one of the receptors for the analyte is attached to a solid support.

In another advantageous aspect, the luminescent tracer compound and the reference compound are 2 distinct compounds excitable at the same wavelength, the reference compound emitting at a wavelength different from that used to measure the amount of analyte .

This second variant of the process of the invention is preferred in particular in the use of a homogeneous process for the detection and / or determination by luminescence of an analyte in a medium capable of containing it in which a reagent coupled with a luminescent tracer compound and a reagent coupled with a heavy atom or motifs containing a heavy atom capable of modulating are used the signal from the luminescent tracer compound. A dosing method of this type is described in application EP 232 348.

In this case, we will use in addition to the luminescent tracer compound coupled to the receptor of the analyte and emitting at a wavelength λ ₂ , another free luminescent compound serving as a reference emitting at a length λ ₁ whose signal is not not modulated by the heavy atom effect and reflects the optical properties of the measurement medium, these 2 compounds being excited at the same wavelength.

Advantageously, this second variant of the method according to the invention is used in a homogeneous method of detection and / or determination by luminescence of an analyte in a medium capable of containing it consisting of:

1) adding to said medium a first reagent consisting of a receptor for said analyte,

2) adding a second reagent chosen from the analyte or at least one of its receptors, one of the two reagents being coupled with a luminescent tracer compound and the other reagent comprising a heavy atom or units containing an atom heavy, as well as a luminescent compound serving as an internal reference,

3) incubating the resulting medium either after the addition of each reagent, or after the addition of the two reagents,

4) to excite the resulting medium, and

5) measuring on the one hand the signal emitted by the luminescent tracer compound, said signal being modulated by the effect of a heavy atom at a wavelength λ ₂ , and on the other hand the signal emitted by the compound of reference to a length λ ₁ .

This second variant of the method in which the luminescent tracer compound and the reference compound are two distinct compounds is also advantageously usable in homogeneous methods for detecting and / or determining a analyte in a medium capable of containing it by methods by excess or by competition involving a donor / acceptor system, as described above for the first variant of the method of the invention.

However, in the case of the second variant, it is necessary to add a luminescent compound serving as internal reference during one of the steps of adding the reagents.

The signal of the reference luminescent compound will then be measured at a wavelength λ ₁ and the signal resulting from the transfer of energy at a wavelength λ ₂ , this measurement then being able to be corrected by the measurement carried out at λ ₁ .

In the present description, the following definitions are defined:

- "analyte" any substance or group of analogous substances to be detected and / or determined;

- "receptor" any substance capable of specifically binding to a site of said analyte;

- "heavy atom" an atom with a high atomic number and whose presence near a fluorescent molecule is capable of inducing an increase in the spin-orbit coupling thereof. As examples of suitable heavy atoms, mention may in particular be made of halogen atoms, mercury, thallium, lead. money;

- "motif containing at least ur. heavy atom" any chemical substance naturally containing at least one heavy atom or on which at least one heavy atom can be attached.

In a preferred aspect, the measurement medium is a biological medium such as a serum medium.

As reference luminescent compounds and / or usable as tracer compounds, fluorescent compounds will advantageously be used.

In particular, use will advantageously be made of a fluorescent compound such as a chelate or a rare earth cryptate, in particular a chelate or a cryptate of terbium, europium, dysprosium, samarium or neodynium. Preferably, a terbium or europium cryptate will be used.

In detection and / or determination methods by fluorescence using the measurement method of the invention, a rare earth cryptate described in patent applications EP 180 492 and 321 353 will advantageously be chosen.

Preferably, terbium cryptate: Tb trisbipyridine or europium cryptate: Eu trisbipyridine, as described in application EP 180 492 or the cryptates Eu trisbipyridine diamine and Tb trisbipyridine diamine described in application EP 321 353, will be used.

In an advantageous aspect, the donor fluorescent compound is a europium cryptate and the fluorescent acceptor compound is chosen from allophycocyanin, allophycocyanin B, C phycocyanin or R phycocyanin.

One can also use as reference luminescent compound or as donor tracer compound, a phosphorescent compound such as eosine or erythrosine. In this case, a fluorescent acceptor compound chosen from chlorophylls such as those cited in applications EP 71 991 and EP 314 406, or porphyrins as cited in application EP 71 991 or phthalocyanines such as those of international application WO 88 04777.

In the case of an assay in a liquid medium using phosphorescent donor compounds, the reading will be carried out either on a solid support, or by adding oxygen-sensing molecules to the measuring medium, these techniques being known to those skilled in the art .

Chlorophylls and phthalocyanines can also be used as fluorescent acceptor compounds using as donor compound a cryptate or a chelate of europium.

In a preferred aspect, the luminescent tracer compound and / or the reference luminescent compound have a lifetime greater than one microsecond.

As a light source allowing the excitation of the luminescent tracer and reference compounds, a modular light source such as those described in Lakowicz, Principles of fluorescent spectroscopy, will advantageously be used,

Plenum Press, New York, 1983, p. 96-100. The measurement method of the invention finds in particular an important application in immunoassays by fluorescence, both in assay methods called by competition and by excess, described in the prior art (Landon, Ann. Clin. Biochem, 1981, 18, 253 and E. SOINI et al. Clin. Chem. 1979, 25, 353).

In particular, the measurement method of the invention can be advantageously used in immunoassays in serum medium.

A further aspect of the invention relates to a device for implementing the measurement method according to the invention.

This device is characterized in that it comprises an excitation light source, means for collecting the light beam emitted following said excitation and means for allowing the measurement of luminescence at two distinct wavelengths.

Advantageously, this device also comprises means for dividing the beam emitted following the excitation.

Such a device is illustrated, by way of example, in FIG. 1.

Figure 1 shows:

- in 1, an excitation light source;

- At 2, a lens focusing the exciting beam towards a filter 3 selecting the wavelength desired for the excitation;

- A dichroic filter 4 placed at 45 reflecting the ultraviolet rays and transmitting visible light, which reflects the exciting beam on a lens 5, the latter focusing it on the microplate 6 containing the samples;

- In 7, a lens collects in combination with the lens 5 the luminescence emission resulting from the excitation and projects it on a dichroic filter 8 which divides the emitted light beam;

the filters 9 and 10 make it possible to select the signals emitted by the reference compound and the tracer compound, before their arrival on photomultipliers 11 and 12. The invention will be better understood with the aid of the examples below which have no limiting character.

EXAMPLE 1:

Correction of fluorescence at 665 nm by detection of an addition of terbium cryptate (trisbipyridine diamine (Tb ³⁺ )) in a homogeneous immunofluorescent assay of prolactin.

This example is carried out on the detection of a standard range of antigen and on the assay of 5 serum samples of unknown prolactin concentration.

METHODOLOGY :

In this assay, the fluorescent donor compounds used are europium cryptate trisbipyridine diamine (Eu ³⁺ )

(tracer) and reference compound terbium cryptate trisbipyridine diamine (Tb ³⁺ ) prepared as described in application EP 321 353 (examples 3 and 4).

As the fluorescent acceptor compound, allophycocyanin (Cyanotech, USA) is used.

Monoclonal anti-prolactin E ₁ and 3D3 antibodies are used (CIS bio international, France) recognizing 2 distinct prolactin epitopes. The preparation of the antibodies labeled with europiumtrisbipyridine diamine cryptate (Eu ³⁺ ) or with llophycocyanin is described below:

Or. Uses the following abbreviations:

APC = allophycocyanin

DTT = dithiothreitol

EuTBP = europium cryptate trisbipyridine diamine (Eu ³⁺ )

HSA = human albumin serum

IgG = immunoglobulin G

SPDP = N-succinimidyl 3 (2-pyridyldithio) propionate

Sulfo-SMCC = sulfosuccinimidyl 4 (n-maleimidomethyl) cyclohexane

1-carboxylate.

1) PREPARATION OF IgG E ₁ -APC

a) Activation of APC by sulfo-SMCC

APC (3 mg) commercially available as precipitated in a 60% solution of ammonium sulfate, is centrifuged. After removal of the supernatant, the residue is taken up in 250 μl of 100 mM phosphate buffer, pH 7.0, then filtered at 0.8 μm in order to remove any particles in suspension.

The filtrate is purified by exclusion chromatography on a G25 superfine column (Pharmacia, Sweden) in the same buffer.

The concentration of APC eluted in the exclusion volume is determined at 650 nm, considering a ε _650nm of 731000M ^-1 CM ^-1 .

Activation of the APC is carried out by adding a sulfo-SMCC solution prepared extemporaneously at a rate of 6.9 mM in a 100 mM phosphate buffer pH 7.0 and allowing the reaction to take place for one hour, at room temperature. , with gentle stirring (molar ratio of 15 to 75 sulfo-SMCC by APC). The APC-maleimide is then purified on a G25 superfine column in 100 mM phosphate buffer, 5 mM EDTA, pH 6.5 and stored at 4 ° C. before coupling on 3D3 IgG.

b) Activation of IgG E ₁ by SPDP

Simultaneously, 5 mg of IgG E ₁ at a rate of 10 mg / ml in a 100 mM phosphate buffer, pH 7.0 are activated by the addition of a solution of SPDP (Pierce, USA) at a rate of 6.4 mM in dioxane in a molar ratio of 4 to 16 SPDP per IgG E ₁ .

After 35 min of activation at room temperature, the IgG pyridine-2 thione is purified on a G25 superfine column in a 100 mM phosphate buffer, 5 mM EDTA, pH 6.5.

The proteins are concentrated and the 2-pyridyl sulfide groups are reduced by a solution of DTT (Sigma, USA) having a final concentration of 19 mM for 15 min at room temperature. DTT and pyridine-2-thione are eliminated by purification on a G25 superfine column in 100 mM phosphate buffer, 5 mM EDTA, pH 6.5. The IgG-SH concentration is determined at 280 nm with an ε _280nm of _210,000 M ^-1 cm ^-1 .

c) Conjugation of IgG E ₁ -SH with APC-maleimide

The fixing of the thiol groups on the maleimides is carried out by adding 2.51 mg of activated APC per mg of IgG E ₁ -SH. After 18 hours of incubation at 4ºC and in the dark under gentle stirring, the thiol functions which remain free are blocked by the addition of a 100 mM solution of N-methyl maleimide

(Sigma, USA) having a final concentration of 20 mM for one hour at room temperature.

The reaction medium is purified by gel filtration on a semi-preparative TSK G3000SW column (Beckmann, USA) in 100 mM phosphate buffer pH 7.0.

The APC and IgG E ₁ concentrations of the purified conjugate, eluted in the first peak, are determined by the absorptions at 280 nm and at 650 nm, according to the following calculation:

[APC] _{Mole / l} = A _650nM / 710000

[IgG] _Mole / l = (A _280nm -A ' _280nm ) / ₂₁₀₀₀₀

with A ' _280nm being the contribution to this wavelength of APC-maleimide.

Human serum albumin (HSA) is added up to 1 g / l to the conjugate which is then divided into aliquots and then frozen at -20ºC.

2) PREPARATION OF IgG 3D3 CONJUGATES - Eu TBP

The preparation of IgG 3D3-SH is carried out according to the protocol described above for IgG E ₁ but with a molar ratio of 7.5 SPDP per IgG 3D3.

To 5 mg (5 10 moles) of Eu TBP is added a 25 mM solution of sulfo-SMCC, in 20 mM phosphate buffer, 10% dimethylformamide (v / v pH 7.0 in a proportion of 2.5 moles of activator per mole of EuTBP.

After 45 min of activation at room temperature, the reaction medium is filtered at 0.8 μm in order to remove the precipitate which may have formed. Undesirable reaction products (sulfo-SMCC, N-hydroxysuccinimide, (N-maleimidomethyl) carboxylic acid) are eliminated by ion exchange chromatography on a Mono Q column (Pharmacia, Sweden) in 20 mM phosphate buffer dimethylformamide 10% (v / v ), pH 7.0 under NaCl shock. The concentration of maleimide Eu TBP is determined at 307 nm with an ε _307nm of 25000 M ^-1 cm ^{-1 as} well as the ratio A _307nm / A _280nm .

In a similar way to that described above we do react the maleimide functions with the thiol functions attached to the antibody, in molar proportions varying from 10 to 30 Eu

TBP maleimide by IgG 3D3-SH.

After 18 hours of incubation at 4 ° C and blocking of the thiol groups (possibly remained free) by

N-methylmaleimide, the uncoupled Eu TBP is removed by dialysis in 100 mM phosphate buffer pH 7.0 at 4 ° C until exhaustion (more fluorescence in the dialysis baths).

The characteristics of the conjugate are determined by its absorptions at 307 nm and at 280 nm using the following values taking into account the inherent absorption of the cryptate determined by the ratio A _307nm / A _280nm .

Eu TBP-maleimide:

ε _307nm = 25000 M ^-1 cm ^-1

At _307nm / At _280nm : determined experimentally.

IgG 3D3-SH:

ε _280nm = _210,000 M ^-1 cm ^-1

ε _307nm = 0 M ^-1 cm ^-1

3) In strips of 12 wells (Microstrip Labsystem 0y, Finland) are successively added:

- 100 μl of standard (prolactin standards from the ELSA-PRL kit, (CIS Bio international) in newborn calf serum) or 100 μl of sample to be assayed (human serum),

- 100 μl of anti-prolactin 3D3 monoclonal antibody, labeled with europium cryptate trisbipyridine diamine (Eu ³⁺ ), at a concentration of 0.5 g / ml of antibody in 100 mM phosphate buffer, 150 mM NaF, BSA (bovine serum albumin) 1 g / l, pH 7.0,

- 50 μl of anti-prolactin E ₁ monoclonal antibody labeled with allophycocyanin at a concentration of 7 μ g / ml of antibody in 100 mM phosphate buffer, 150 mM NaF, BSA 1 g / l, PH 7.0,

- 50 μl of terbium cryptate trisbipyridine diamine (Tb ³⁺ ) 10 ^-7 M in 100 mM phosphate buffer, 150 mM NaF, 1 g / l BSA, pH 7.0. After an hour of incubation at 37ºC, the fluorescence of each well is measured according to two protocols:

In a first protocol, fluorescence at 665 nm is detected in resolved time, with a measurement delay of 50 μs and an integration time of 400 μs. The duration of the measurement is 1 s.

The excitation is caused by a flash lamp, pulsed at 1000 Hz.

The excitation wavelength is centered by an interference filter at 307 nm, maximum absorption of trisbipyridine diamine

(Eu ³⁺ ) and trisbipyridine diamine (Tb ³⁺ ). The fluorescence intensity noted, reflecting the energy transfer, is proportional to the concentration of prolactin antigen present in the incubation medium.

The measurement is carried out using an ARCUS fluorimeter

(LKB, Sweden) using an interference filter suitable for the emission of the fluorescent acceptor compound and the reference compound.

The measurement can also be carried out in a single step using the prototype fluorimeter described in Example 2. In this case, a white 96-well microplate (Dynatech, USA) will be used for the measurement.

The standard curve FLUO _665nm = f ([Prolactin]) is plotted and the concentration of the 5 samples is measured.

The results are reported in Table I below.

TABLE I

Standard ELSA-PRL [Prolactin] μUI / ml

STD ₀ 0

STD ₁₁₆₅

STD ₂₃₀₀

STD ₃₉₂₀

STD ₄ 3100

STD ₅ 6500

Dosing serum [Prolactin] (FLUO ₆₆₅ ) μUI / ml Ech1 141

Ech2 951

Ech3 113

Ech4 287

Ech5 699 In a second protocol, the fluorescence at 665nm is measured in the same way as described above. However, a second measurement of the same well is carried out at 545nm after a second excitation, under the same time reading parameters (delay, integration time and duration of the measurement). The fluorescence emission thus measured in time resolved at 545 nm is characteristic of the emission of trisbipyridine diamine (Tb ³⁺ ) used as an internal reference and reflects the optical properties of the medium to be assayed.

The standard curve FLUO _665nm / FLU0 _545nm = f ([Prolactin]) is plotted and the concentration of the 5 samples is measured. The results are reported in Table II below:

TABLE II

Standard ELSA-PRL [Prolactin] μUI / ml

STD ₀ 0

STD ₁₁₆₅

STD ₂₃₀₀

STD ₃₉₂₀

STD ₄ 3100

STD ₅ 6500

Dosing serum [Prolactin] (FLUO ₆₆₅ / FLUO ₅₄₅ ) μUI / ml

Ech1 580

Ech2 428

Ech3 417

Ech4 71

Ech5 2044

Finally, as a reference, the 5 samples are also assayed by the ELSA-PRL radioimmunometric assay kit (CIS bio international, France).

The comparisons of the assay results carried out on the five samples are collated in Table III below:

TABLE III [prolactin] μUI / ml

ELSA-PRL FLUO ₆₆₅ FLUO ₆₆₅ / FLUO ₅₄₅

Ech1 612 141 580

Ech2 395 951 428

Ech3 425 113 417

Ech4 59 287 71

Ech5 1870 699 2044 These results show that the measurements of the concentrations of the prolactin samples by the method comprising a correction of the fluorescence intensity (FLUO ₆₆₅ / FLUO ₅₄₅ ) are in good correlation with the results obtained using the ELSA radioimmunometric assay. PRL, whereas this is not the case when the concentration is measured only by the fluorescence intensity at 665nm without correction (FLUO ₆₆₅ ).

EXAMPLE 2:

Correction of the fluorescence at 665nm by detection of the fluorescence of the antibody-cryptate conjugate at 620nm in a homogeneous immunofluorescent assay of prolactin.

The fluorescence measurement is carried out using a prototype fluorimeter, which is described below:

An N2 VSL 337 laser (LSI, USA) is used as an excitation source (wavelength at 337 nm). The pulse duration is specified at 3 nanoseconds and is repeated at a frequency of 10 Hertz. The beam passes through a filter (CORNING) in order to eliminate any stray light at excitation other than 337 nm.

After entering the measurement chamber, the beam is reflected by a dichroic filter, placed at 45 degrees, which has the property of reflecting ultraviolet light and of being able to transmit visible light.

The beam reflected by the dichroic filter is focused on the well to be measured of a microplate by a lens of fused silica. The fluorescence emission is collected at a solid angle of 20 degrees, neck bounded by the same lens, and passes directly through the dichroic filter (fluorescence in visible light).

An interference filter, with characteristics defined according to the fluorescence wavelength to be detected, makes it possible to get rid of the lights that can interfere with the signal, the intensity of which is then measured by a photomultiplier

(HAMAMATSU R2949).

The photon counter used is an SR-400 (STANFORD RESEARCH SYSTEMS), whose operations and synchronization with the laser are controlled by an IBM PC-AT type computer via an RS 232 output. The pulses from the photomultiplier are recorded during a time window (t _g ) and after a delay (t _d ) determined provided that they are greater than a discriminating level selected by the photon counter in order to optimize the signal / noise of the photomultiplier.

An XY table, controlled by the IBM PC-AT, allows the different positioning of the measurement microplate by stepping motors, including the loading, positioning under the exciting beam, automatic sequential reading of the 96 wells , and exit.

I. Variation of the signal of a cryptate conjugate at 620nm depending on the serum.

In a white 96-well microplate (Dynatech, USA) are introduced:

- 100 μl of serum to be tested

- 200 μl of an anti-prolactin-europium cryptate antibody conjugate at a concentration of 0.5 μg / ml of antibody in 100 mM phosphate buffer pH 7.5, HSA 1 g / l, 150 mM NaF.

The europium cryptate used is trisbipyridine diamine (Eu ³⁺ ), the preparation of which is described in application EP 321 353 (examples 3 and 4).

The anti-prolactin antibody used is the 3D3 antibody (CIS bio international, France).

The 3D3 antibody-europium cryptate antibody conjugate is prepared as described in Example 1.

After 1 hour of incubation at 37 ° C, fluorescence is detected at 620nm, in resolved time, with a measurement delay of 50 μs and an integration time of 400 μs. The measurement time is 1 s.

The optical density of the serum is measured at 310 nm using a Lambda 15 spectrophotometer PERKIN ELMER (UK). The results are reported in Table IV below.

TABLE IV

Serum Do at 310 nm Signal at 620 nm 1 0.66 25 693

2 3.3 7 754

3 0.33 33 156

These results show that the measured signal varies greatly as a function of the optical density of the serum.

Under these conditions, it is impossible to carry out a reliable assay because the result does not only depend on the amount of analyte in the serum but also on the optical quality of the serum.

II. Correction of the acceptor's signal.

An immunoassay is then carried out by successively adding to the microplate:

- 100 μl of serum

- 100 μl of anti-prolactin monoclonal antibody conjugate

3D3-europium cryptate trisbipyridine diamine (Eu ³⁺ ) at the concentration of 0.5 IU / ml of antibody in the phosphate buffer above,

- 100 μl of monoclonal anti-prolactin E ₁ -allophycocyanin conjugate at a concentration of 3.5 μg / ml of antibody in the same buffer.

This antibody conjugate E ₁ (CIS bio international France) -allophycocyanin (Cyanotech, USA) is prepared as described in Example 1.

The measurement is carried out at 665nm and the measured value is corrected by dividing this value by the value of the fluorescence measured at 620nm which reflects the optical properties of the medium.

The concentration values determined using a standard curve as indicated in Example 1, with or without correction, are compared with the values obtained using as reference the ELSA-PRL radioimmunometric assay kit (CIS bio international France).

The results are reported in Table V below, in which the values of the prolactin concentrations expressed in pu / ml are determined respectively by the ELSA-PRL assay (ELSA) and by the measurement of fluorescence at 665nm uncorrected (FIA ₆₆₅ ) and corrected (FIA _665/620 ), as well as the value of the fluorescence measured for the different samples at 620nm (FLUO ₆₂₀ ) expressed in counts per second.

TABLE V

Sample no ELSA FIA _665/620 FIA ₆₆₅ FLUO ₆₂₀ 1 358 394 195 42 148

2,530,592 91 28,272

3 65 92 18 40 495

4,153,178 94 43,363

5,251,289 86.7 38,007

6,161,146 86.4 44,531

7,179 189 77 40,873

8,285,297 161 41,403

9,310,330 106 35,214

10 2744 3427 1470 31670

11 196 153 15 36 551

12,244 198 55 38,134

13 447 477 300 40 340

14,251,234,350 58,615 These results confirm the variability of the measured signal as a function of the serum sample assayed.

Indeed, since the antibody-cryptate conjugate is in excess in the assay relative to the analyte sought, the signal measured at 620nm (which corresponds to the conjugate not engaged in the complex) should be constant. In addition, we note that the prolactin concentration values determined from the measurement of the signal at 665nm (FIA ₆₆₅ ) do not correspond to the values obtained by the reference assay, while the values obtained by the corrected measurement (FIA _{665 / 620} ) are in good correlation with these.

This correlation is illustrated by the curves of FIGS. 2 and 3, on which the prolactin concentration determined by the ELSA-PRL test in IU / ml is represented on the abscissa and, on the ordinate, respectively, the concentration determined by the corrected measurement FIA _{665 / 620} (Figure 2), or by the uncorrected FIA ₆₆₅ measurement (Figure 3), expressed in IU / ml.

The results obtained on 97 sera show that the correlation is only obtained with the correction of the signal measured at 665nm.

Claims

1. Method for measuring the luminescence emitted in a luminescence assay, characterized in that at least one luminescent tracer compound and a luminescent compound used as internal reference are used which, when subjected to the same length d excitation wave, are capable of emitting either by direct luminescence or by induction of an emission of luminescence, at different wavelengths, respectively λ ₂ and λ ₁ , and in that the measurement is corrected of the luminescence emitted by the tracer compound at the wavelength λ ₂ by measuring the luminescence emitted by the reference compound at the wavelength λ ₁ .

2. Method according to claim 1, characterized in that the luminescent tracer compound and the reference compound are one and the same compound.

3. Method according to claim 1, characterized in that the luminescent tracer compound and the reference compound are distinct.

4. Method according to any one of claims 1 to 3, characterized in that the luminescent tracer compound and / or the reference compound are fluorescent compounds.

5. Method according to any one of claims 1 to 4, characterized in that the luminescent tracer compound and / or the reference compound are chelates or rare earth cryptates.

6. Method according to any one of claims 1 to 5, characterized in that the luminescent tracer compound and / or the reference compound have a lifetime greater than one microsecond.

7. Method according to any one of claims 1 to 6, characterized in that one simultaneously performs the measurement of the luminescence emitted by the reference compound and by the tracer compound, respectively, at wavelengths λ ₁ and λ ₂ .

8. Use of the method according to claim 1 in a homogeneous method of detection and / or determination of an analyte in a medium capable of containing it.

9. Use according to claim 8, in a homogeneous method for detecting and / or determining an analyte in a medium capable of containing it using an excess method consisting of:

4) to excite the resulting medium at the excitation wavelength of the luminescent donor compound, 5) to measure the signal of the luminescent donor compound at a wavelength λ ₁ , this measurement serving as a reference, and the resulting signal energy transfer at a wavelength λ ₂ .

10. Use according to claim 8, in a homogeneous method of detection and / or determination of an analyte in a medium capable of containing it using a method by competition consisting of:

1) adding, to said medium containing the desired analyte, a first reagent consisting of a receptor for said analyte, coupled with a luminescent donor compound,

11. Use according to claim 8, in a homogeneous method for detecting and / or determining an analyte in a medium capable of containing it using a competitive method consisting of:

1) adding, to said medium containing the desired analyte, a first reagent consisting of a receptor for said analyte, said receptor being coupled with a luminescent acceptor compound,

5) measuring the signal of the donor luminescent compound at a wavelength λ _1, this measurement serving as a reference, and the signal resulting from the transfer of energy at a different wavelength λ ₂ .

12. Use according to claim 8, in a homogeneous method for detecting and / or determining an analyte in a medium capable of containing it using a method consisting of:

3) incubating the resulting medium either after the addition of each reagent or after the addition of the two reagents,

4) to excite the resulting medium, and

5) measuring on the one hand the signal emitted by the luminescent tracer compound, said signal being modulated by the effect of a heavy atom at a wavelength λ ₂ r and on the other hand the signal emitted by the compound of reference to a length λ ₁ .

13. Use according to any one of claims 9 to 12, characterized in that the luminescence emitted by the reference compound and by the tracer compound is measured simultaneously, at wavelengths λ1 and λ2, respectively.

14. Use according to any one of claims 9 to 11, characterized in that at least one of the analyte receptors is attached to a solid support.

15. Device for implementing the method according to claim 1, characterized in that it comprises a source of light excitation, means for collecting the light beam emitted following said excitation, and means for enabling luminescence measurement at two distinct wavelengths.

16. Device according to claim 15, characterized in that it also comprises means for dividing the beam emitted following the excitation.

17. Device according to one of claims 15 or 16, characterized in that it also comprises a method of correcting the measurement carried out at the wavelength λ2 integrated into the apparatus, consisting in fixing a counting rate on the channel measuring the luminescence emission of the reference compound at the wavelength λ1, then, when this rate is reached, triggering the end of the measurement on the channel measuring the luminescence emission at the wavelength λ2.